1. Field of the Invention
This invention is directed to tools, compositions and methods for identifying genetic mutation and mega-bases of nucleic acid information by sequencing and, in particular, to electronic media and programs for analyzing sequences, genes and complete genomes by sequencing, and to the mutations identified and kits comprising reagents for identifying mutations in biological samples.
2. Description of the Background
Mycobacterium tuberculosis (MTB), the causative agent for tuberculosis, is a highly transmissible bacterial pathogen with significant morbidity and mortality, particularly in HIV infected patients. Since 1997 tuberculosis has remained the leading cause of death in South Africa, a statistic linked to this country's growing HIV epidemic. Moreover, effective treatment measures in patients with active MTB have been exacerbated by increasing cases of multidrug resistance (MDR) and extensively drug-resistant (XDR) clinical isolates.
Microscopy remains the cornerstone for diagnosing MTB in many low resource areas of the world where both MTB and also HIV are prevalent. However, many HIV infected patients with MTB are smear negative and microscopy provides no information about antibiotic resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant strains (XDR) has rendered standard MTB treatment regimens ineffective. According to one study, approximately 20% of TB patients in South Africa with HIV have MDR MTB. Rapid detection of MTB and initiating effective therapy is critical to decrease transmission and improve treatment outcome. The roll-out of Cephiad's Gene Xpert (Xpert) has improved MTB diagnosis and provides evidence of Rifampin resistance, but information about other drugs is not provided. Furthermore, it may not be feasible to place Xpert testing in many microscopy labs in low resource settings. The ability to efficiently ship sputum samples centrally for next-generation sequencing (NGS) offers an opportunity to utilize highly trained staff and available infrastructure at central or regional laboratories.
MDR tuberculosis strains are resistant to the first line antibiotics rifampin (RIF) and isoniazid (INH), while XDR MTB strains are resistant to both RIF and INH as well as any fluoroquinolone and second-line injectable antibiotic drugs (e.g., amikacin, kanamycin or capreomycin). About 6% of all MTB cases are MDR strains and South Africa continues to report higher percentages of XDR cases each year. While 7% of patients infected with standard MTB strains succumb to infection, the death rate rises to almost 50% with MDR tuberculosis. The emergence of antibiotic resistant MTB strains underscores an immediate need for rapid and highly accurate diagnosis, particularly in the developing countries of Africa. In addition migratory populations make geographical surveillance and tracking of drug resistance strains more urgent.
Culture-based drug susceptibility testing (DST) for MDR strains is considered the gold-standard, but is time consuming (weeks to months), technically challenging and cost prohibitive, especially in resource limited countries. For example, the BACTEC MGIT 960 (Becton Dickinson Microbiology System, Silver Sparks Nev., USA), is an automated continuously culture-based monitoring system that measures bacterial oxygen consumption and can perform DST using prepared kits which are available for susceptibility of strains to a number of antibiotics. DST results obtained with the BACTEC MGIT 960 yield reliable and reproducible but require handling of viable and potentially infectious cultures, days to weeks or delay until results are available, specialized laboratory accommodations and high costs associated with the instrument and consumables.
In recent years, several nucleic acid based assays for determining MTB drug resistance have been developed. One of the most popular commercially available diagnostic assays is the GenoType MTBDRplus Line Probe Assay (LPA) by Hain LifeScience. This test employs nucleic acid extraction, PCR amplification, probe hybridization and colorimetric visualization on lateral strips via an alkaline phosphatase reaction. LPA has been shown to be sensitive and specific, but there are several drawbacks. Sensitivity of the LPA for all resistance-associated mutations will most likely never reach 100% since many mutations that confer resistance have yet to be discovered. Another inherent limitation of the LPA is an inability to detect sample populations that contain a mixture of resistant and susceptible strains. Strains that harbor substitution mutations that change an amino acid to a previously uncharacterized or unknown mutation not presented on the LPA are not detected. Furthermore, the LPA only allows detection of the most frequent mutations that cause resistance. If a strain were to contain mutations outside of the targeted mutations, the wild-type banding pattern will appear leading to a false negative (susceptible) result.
Thus, there is a need for a rapid, standardized, cost-effective protocol for full length gene analysis of critical genes such as, for example, genes associated with first and second line drug resistance.